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Background: Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines., Results: L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding alphavbeta5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies., Conclusion: Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain.

Regulation of MUC1 expression and removal is a salient feature of embryo implantation, bacterial clearance, and tumor progression. In some species, embryo implantation is accompanied by a transcriptional decline in uterine epithelial expression of MUC1. In other species, MUC1 is locally removed at blastocyst attachment sites, suggesting a proteolytic activity. Previously, we demonstrated that MUC1 is proteolytically released from the surface of a human uterine epithelial cell line, HES, and identified TNFalpha converting enzyme/a disintegrin and metalloprotease 17 as a constitutive and phorbol ester-stimulated MUC1 sheddase. The aims of the current study were to test the ability of soluble factors elevated during the periimplantation interval in vivo to stimulate ectodomain shedding of MUC1 from HES uterine epithelial cells and to characterize the nature of this proteolytic activity(ies). We identified TNFalpha as a prospective endogenous stimulus of MUC1 ectodomain release and of MUC1 and TNFalpha converting enzyme/a disintegrin and metalloprotease 17 expression. Moreover, we established that TNFalpha-stimulated MUC1 shedding occurs independently of increased de novo protein synthesis and demonstrated that the TNFalpha-induced increase in MUC1 gene expression is mediated through the kappaB site in the MUC1 promoter. Finally, we determined that the TNFalpha-sensitive MUC1 sheddase is inhibited by the metalloprotease inhibitor, TNFalpha protease inhibitor (TAPI), and the endogenous tissue inhibitor of metalloprotease-3. Collectively, these studies provide the initial in vitro characterization of a putative physiological stimulus of MUC1 ectodomain release and establish the nature of the metalloproteolytic activity(ies) involved.

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in human breast cancers. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. We demonstrate that MUC1 expression in T47D breast cancer cells and normal human mammary epithelial cells (HMEC) is enhanced by tumor necrosis factor-alpha (TNF-alpha) in the presence of interferon-gamma (IFN-gamma). MUC1 responsiveness to these cytokines was modest in T47D cells and robustly induced in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that a kappaB site at -589/-580 and the STAT-binding element at -503/-495 and were required for cooperative stimulation by TNFalpha and IFN-gamma. Binding of NFkappaB p65 to the MUC1 kappaB site was induced by TNF-alpha treatment, as demonstrated by electrophoretic mobility shift assay. Specific mutation of the kappaB site prevented binding of NFkappaB p65 and blocked TNF-alpha stimulation of MUC1 promoter activity. Collectively, these studies demonstrate synergistic stimulation of MUC1 expression by TNF-alpha and IFN-gamma that is mediated by independent actions of NFkappaB p65 and STAT1alpha upon kappaB and STAT sites, respectively, in the MUC1 promoter. Strong induction of MUC1 expression by these proinflammatory cytokines is clearly evident in normal mammary epithelium. In contrast, breast tumor cells appear to override normal regulatory responses via as yet undefined cis-elements., (Copyright 2002 Wiley-Liss, Inc.)